High-frequency Triazole Resistance Found In Nonculturable Aspergillus fumigatus from Lungs of Patients with Chronic Fungal Disease
High-frequency Triazole Resistance Found In Nonculturable Aspergillus fumigatus from Lungs of Patients with Chronic Fungal Disease
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DOI：
10.1093/cid/cir179
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发表时间：
2011-05-01
影响因子：
11.8
通讯作者：
Perlin, David S.
中科院分区：
文献类型：
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作者：
Denning, David W.;Park, Steven;Perlin, David S.
背景口服三唑类药物治疗侵袭性（IPA）、过敏性（ABPA）和慢性肺曲霉病（CPA）已得到广泛认可，且通常为长期治疗。三唑耐药率在国际上不断上升。曲霉菌病的微生物诊断受到培养产量低的限制，导致三唑耐药频率的不确定性。使用超灵敏实时聚合酶链反应（PCR）检测曲霉属，我们评估了支气管肺泡灌洗（BAL）和痰标本中的呼吸道真菌负荷。在PCR阳性，培养阴性的样本中，我们进一步扩增CYP 51 A基因，以检测与三唑耐药相关的关键单核苷酸多态性（SNP）。正常志愿者BAL中检出曲霉菌DNA（4/11，36.4%），经培养或显微镜证实为IPA的患者BAL中检出曲霉菌DNA（21/22，95%）。ABPA和CPA患者痰中曲霉菌DNA检出率分别为78.9%（15/19）和71.4%（30/42），而培养法分别为0%和16.7%。在培养阴性、PCR阳性的样本中，我们在55.1%的样本中检测到药物靶标CYP 51 A内的三唑耐药突变（L98 H与串联重复序列[TR]和M220）。ABPA患者8例中有6例（75%）和CPA患者24例中有12例（50%）存在耐药标志物，其中一些患者既往未接受过三唑治疗，大多数患者在采样时血浆药物浓度充足。导致感染的真菌的微生物负荷非常低，以前无法直接培养和检测临床样本中的抗真菌药物耐药性。这些发现对三唑类药物用于人类抗真菌治疗的可持续性具有重要意义。
Background. Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance.Methods. Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance.Results. Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling.Conclusions. The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.